Progression of DCIS to invasive breast cancer through CCR2 chemokine signaling

描述(申请人提供)：导管原位癌(DCIS)是女性诊断的最常见的非浸润性乳腺癌类型，是浸润性导管癌(IDC)的直接先兆。目前的细胞和组织病理学方法不能准确地预测疾病的进展，导致患者对DCIS治疗不足或治疗过度。我们的长期目标是确定导致IDC的关键因素，使我们能够开发一种基于分子的方法来预测IDC的风险，并开发一种更定制的方法来治疗DCIS。DCIS中CCL2/CCR2趋化因子受体信号转导可能是DCIS向IDC发展的重要机制。CCR2及其结合配体CCL2在乳腺导管癌中过表达，并与肿瘤分级和患者预后不良有关。CCL2/CCR2趋化因子信号最为人所知的是调节巨噬细胞募集。然而，CCL2的敲除显著地抑制了乳腺肿瘤的进展，而不显著地影响巨噬细胞的募集。CCR2在导管癌细胞中的过表达增强了细胞的存活率和侵袭力，与促生存和亲侵袭蛋白(ALDH1A1)的表达增加和促凋亡蛋白(Htra2)的表达减少有关。这些研究表明CCR2信号在乳腺导管癌进展中起着重要作用。为了阐明CCL2/CCR2信号在体内的作用，我们通过小鼠乳腺导管内注射(Mind)人乳腺癌细胞株Sum225和DCIS.com建立了新的异种移植模型。Sum225 Mind模型形成稳定的非侵入性病变，而DCIS病变在DCis.com Mind模型中进展为IDC。与传统的移植模型相比，这些心理模型更接近于在患者身上发现的DCIS的生物学特征。与Sum225相比，DCIS.com病变显示CCL2、CCR2和ALDH1A1蛋白的高表达和Htra2的低表达，这与浸润性癌的进展有关。根据初步研究，我们假设导管癌细胞中的CCL2/CCR2信号促进了DCIS向IDC的进展。目的：利用原代和已建立的乳腺癌细胞系，在思维模型、三维细胞培养模型和生化方法中分析CCR2过表达和下调对导管癌细胞DCIS中自分泌CCL2/CCR2信号转导的影响，以探讨CCL2/CCR2信号在导管癌细胞DCIS中的作用。目的：利用原代和已建立的乳腺癌细胞系，采用肾下移植模型、心理模型、三维细胞培养模型和生化方法，分析成纤维细胞来源的CCL2对DCIS进展的影响，以确定基质来源的CCL2在CCR2介导的DCIS进展中的作用。通过多重PI计划，目的是确定CCL2/CCR2信号如何调节促侵袭和促生存因子(ALDH1A1)和促凋亡因子(Htra2)的表达，从而促进导管癌的进展。这些研究将揭示趋化因子受体信号在DCIS中的作用机制，具有重要的治疗意义。。

DESCRIPTION (provided by applicant): Ductal carcinoma in situ (DCIS) is the most common type of non-invasive breast cancer diagnosed in women and an immediate precursor to invasive ductal carcinoma (IDC). Current cyto- and histopathological approaches do not accurately predict disease progression, resulting in patients being under-treated or over-treated for DCIS. Our long-term goals are to identify key factors that lead to IDC that will enable the development of a molecular based approach to predict the risk of IDC, and a more tailored approach to treat DCIS. CCL2/CCR2 chemokine receptor signaling in DCIS is a potentially important mechanism for DCIS progression to IDC. CCR2 and its binding ligand CCL2, are overexpressed in breast ductal carcinomas, and correlate with tumor grade and poor patient prognosis. CCL2/CCR2 chemokine signaling is best known for regulating macrophage recruitment. However, knockdown of CCL2 significantly inhibits mammary tumor progression without significantly affecting macrophage recruitment. Overexpression of CCR2 in ductal carcinoma cells enhances cell survival and invasiveness, associated with increased expression of pro-survival and pro-invasive proteins (ALDH1A1) and decreased expression of pro-apoptotic proteins (Htra2). These studies indicate an important role for CCR2 signaling in breast ductal carcinoma progression. To clarify the in vivo role of CCL2/CCR2 signaling, we have developed novel xenograft models through mouse Mammary intraductal injection (MIND) of human breast carcinoma cell lines, Sum225 and DCIS.com. The Sum225 MIND model form stable non-invasive lesions, while DCIS lesions progress to IDC in the DCIS.com MIND model. Compared to conventional transplant models, these MIND models more closely mimic the biology of DCIS found in patients. Compared to Sum225 lesions, DCIS.com lesions show higher expression of CCL2, CCR2 and ALDH1A1 proteins and lower expression of Htra2, correlating with progression to invasive carcinoma. Based on preliminary studies, we hypothesize that CCL2/CCR2 signaling in ductal carcinoma cells enhances progression of DCIS to IDC. Aim 1: To characterize the role of autocrine CCL2/CCR2 signaling in ductal carcinoma cells during DCIS by analyzing effects of CCR2 overexpression and knockdown in MIND models, 3D cell culture models and biochemical approaches, using primary and established breast cancer cell lines. Aim 2: To determine the functional contribution of stromal derived CCL2 on CCR2 mediated progression of DCIS, by analyzing the effects of CCL2 derived from fibroblasts on DCIS progression, using subrenal graft transplant models, MIND models, 3D cell culture models and biochemical approaches, using primary and established breast cancer cell lines. Through a multiple PI plan, the objective is to determine how CCL2/CCR2 signaling regulates expression of pro-invasive and pro-survival factors (ALDH1A1) and pro-apoptotic factors (Htra2) to promote ductal carcinoma progression. These studies will reveal mechanistic insight into the role of chemokine receptor signaling in DCIS, with important therapeutic implications. .